FAQs

myTags Expert SARS-CoV-2 probes are available two ready-to-use formats:

• Immortal probes that are ready to be coupled with your preferred tag(s)
• Labeled probes with Alexa-488, ATTO-550, or ATTO-647N fluorescent tags. For experiments that need a brighter signal, the 3X fluorescent tag upgrade is recommended, which can aid detection of lower cellular viral RNA concentrations at earlier experimental time points.

Yes. Due to the manufacturing process, there might be a small pellet visible. It is critical that you resuspend the myTXTL Master Mix completely before aliquoting it to set up your TXTL reaction(s).

Yes, myTXTL Linear DNA Expression Kit allows the use of both, linear and circular/plasmid templates.

Sample handling and storage is mainly determined by the stability of your molecule of interest (protein, DNA, RNA) and thus optimal conditions may need to be evaluated. But to ensure sample integrity, we would recommend to either process the myTXTL reaction immediately after performing the incubation or store it at ≤ -20 °C.

You are advised to keep the number of freeze-thaw-cycles to a minimum. Nevertheless, our studies have indicated that up to five freeze-thaw-cycles do not negatively influence protein production efficiency of the myTXTL Master Mix when using flash freezing in liquid nitrogen.

Yes, please see examples in the Publications section; filter for myTXTL. Please note, that every gene circuit should start with a σ70-specific promoter like P70a.

Most importantly, the excitation and emission wavelength should match the fluorescence properties of deGFP/eGFP (e.g. λEm 488 nm, λEx 535 nm). Other reader settings such as reading mode, integration time and gain value should be chosen under consideration of high well-to-well fluorescence reading reproducibility.

Efficient in vitro protein production is highly dependent on the quality of the template DNA, which should be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery (e.g. EDTA, ethidium bromide, SDS, Cl- ions, ethanol). Preparation of plasmid DNA with standard commercial kits usually involves sample treatment with RNase, which may not be completely removed during downstream processing. Thus, we strongly recommend subjecting the prepared DNA to either a commercial PCR clean-up kit or standard phenol-chloroform extraction and ethanol precipitation. Ideally, template DNA is suspended in nuclease-free water. Please note, introducing Mg2+and K+ ions can compromise the kit performance, as they are extremely critical for transcription and translation, and are optimized in the master mix.

Yes. For all plasmids containing the lambda phage promoter (P70a, P70b, P70c, P70d) it is extremely crucial to use E. coli KL740 as the transformation strain. When cultivated below 30°C, this strain over-expresses the lambda phage repressor protein Cl857 that represses P70 promoters, thus ensuring high transformation efficiency and plasmid stability. KL740 can be purchased from E. coli Genetic Stock Center (Yale) [CGSC#: 4382] or from Daicel Arbor. For all other plasmids, a standard laboratory E. coli cloning strain like JM109 or DH5alpha is sufficient.

No. The myBaits Expert Human Affinities kit is a unique chemistry that should not be coupled with other myBaits Custom or myBaits Expert products. Please contact us for technical questions regarding the use of our products.

The myBais Expert Human Affinities kit protocol is optimized for degraded and ancient DNA samples to support one NGS library per capture reaction (dual-round enrichment). We do not recommend pooling samples without trials.

Yes, Daicel Arbor Biosciences has an NGS services division adept at preparing and working with ancient DNA libraries. Learn more about our myReads NGS laboratory and sequencing service options, and contact us today to discuss your NGS project goals with our team of ancient DNA experts.

Read data acquired with the Human Affinities kit can be used for a wide range of population genetics analysis. However, because this kit exhibits allelic read coverage bias distinct from biases in other assays (like shotgun sequencing and other targeted sequencing kits), we recommend that data generated with the Human Affinities kit be used alone for whatever analyses you may perform.

Daicel Arbor Biosciences is currently in the process of generating a list of sites that are not expected to show allelic coverage bias, and thus can be used in conjunction with the unbiased portion of data from other assays. To be notified if this list becomes available, please Contact Us and request to be added to our Human Affinities product notification mailing list.

The myBaits Expert Human Affinities kit is compatible with virtually any NGS library type that can be amplified with universal primers (before and after capture), including commercial and DIY methods.

myBaits Expert Human Affinities Manual [PDF]

The protocol and reagents provided with the myBaits Expert Human Affinities kit were developed for ancient DNA and other library types that are heavily degraded or contaminated with non-human DNA. It involves two rounds of hybridization capture, with hybridization incubated at high temperature (70°C), with hybrid bead-binding and washes performed at a lower temperature (55°C or room temperature). Each round involves an over-night incubation, and libraries are amplified following both rounds. When enriching non-contaminated/degraded libraries, a single round of enrichment is recommended.

The protocol and reagents provided with the myBaits Expert Human Affinities kit were developed for ancient DNA and other NGS library types that are heavily degraded or contaminated with non-human DNA. It is fully compatible with modern and fresh DNA samples as well.

Yes. Specific recommendations for library pooling for co-enrichment with myBaits Custom Methyl-Seq kits can be found in the applicable myBaits manual.

Hybridization capture is integrated into the overall NGS workflow immediately before sequencing on an NGS platform, such as Illumina. A fully sequenceable, barcoded/indexed NGS library is made from bisulfite or enzymatically-converted DNA. A pool of these multiple barcoded NGS libraries is then denatured, and allowed to anneal to complementary target-specific biotinylated probes/baits. These bait:library complexes are then bound to streptavidin-coated magnetic beads via the biotin on the probes, which are washed to remove non-specifically bound molecules. The remaining “enriched” library molecules are then released from the baits and amplified before sequencing.

Note! You may know the “hybridization capture” technique by another name, such as:

• Target enrichment
• Target capture
• Probe capture
• Exon capture
• Capture sequencing / sequence capture
• Hybridization sequencing / hyb-seq

Use myBaits Custom Methyl-Seq with PCR-amplified and amplifiable NGS libraries generated from bisulfite- or enzymatically-converted nucleic acids in which non-methylated cytosines have been converted to uracils, and following PCR amplification these positions are now thymines. Compatible formats include Illumina® TruSeq®-style, Illumina Nextera® Flex-style, Ion Torrent®, or other libraries with universal adapter priming sites. Do NOT use myBaits with PCR-free libraries; additionally, myBaits are incompatible with libraries made using original Nextera or Nextera XT library preparation kits, or any library type containing biotin. Dual-indexed libraries are strongly recommended to reduce the hazard of mis-indexing induced by PCR jumping events. The applicable myBaits manual provides detailed protocol instructions for enriching libraries intended for methylation sequencing.

If you are using a never-before-tried library prep protocol to pair with your myBaits kit, we recommend that you first perform some total library (shotgun) sequencing before doing myBaits enrichment. This is important in order to verify that your chosen library prep protocol/kit generates libraries of sufficient complexity and minimal bias in your hands, otherwise you may experience poor target capture results. High quality libraries are absolutely essential for achieving a successful target capture project.

Use myBaits with PCR-amplified and amplifiable NGS libraries, including Illumina TruSeq® -style, Illumina Nextera® Flex-style, Ion Torrent, or other libraries with universal adapter priming sites. It is NOT recommended to use myBaits with PCR-free libraries. Additionally, myBaits are incompatible with libraries made using original Nextera or Nextera XT library preparation kits, or any library type containing biotin. Dual-indexed libraries are strongly recommended to reduce the hazard of mis-indexing induced by PCR jumping events. The applicable myBaits manual provides detailed protocol instructions for enriching libraries for sequencing on short- and/or long-read platforms (e.g. PacBio® or Oxford Nanopore Technologies®).

If you are using a never-before-tried library prep protocol to pair with your myBaits kit, we recommend that you first perform some total library (shotgun) sequencing before doing myBaits enrichment. This is important in order to verify that your chosen library prep protocol/kit generates libraries of sufficient complexity and minimal bias in your hands, otherwise you may experience poor target capture results. High quality libraries are absolutely essential for achieving a successful target capture project.

Provided below are a list of companies that sell NGS library prep kits that are known to be compatible with myBaits. This is NOT an exhaustive list; there are many other unlisted options that are also compatible with myBaits. Also, kits on this list may not necessarily be appropriate for your samples. NGS library prep is not “one size fits all”; different factors such as sample type, DNA input amount, genome complexity, and sequence composition may influence the type of library prep kit that would be best for your application. For example, low input, degraded, and/or damaged DNA templates may require special handling (see below) and/or modifications to commercial kits.

Contact these and other manufacturers to learn about your options and find what works best for your samples and project needs:

• Biosearch / Lucigen
• Claret Bioscience
• Illumina
• New England Biolabs
• Kapa Biosystems
• PerkinElmer / Bioo Scientific
• Rubicon Genomics / Takara
• Swift Biosciences / IDT

To produce a Custom WGE panel, a high-purity, high-molecular weight genomic DNA (gDNA) sample is required to be shipped to Arbor’s manufacturing facility in Ann Arbor, MI, USA.

If you do not have access to the appropriate gDNA, for some organisms Arbor may be able to provide the additional service of acquiring the gDNA sample(s) from a third-party vendor.

Custom gDNA sourcing service is only upon prior arrangement and approval by Arbor. This service is only available for species whose genomic DNAs are available from vendors supplying the US. The gDNA sample(s) must be available for Arbor to purchase in sufficient quantity as a commercial entity without any special licenses, acquisition or use permits, or memberships.

Please contact us to provide specific details on the gDNA source(s)/species that you are interested in, and we will review the request to determine feasibility and pricing.

Target capture necessarily requires subjecting your libraries to a bottleneck, wherein target molecules are captured and enriched, and non-target molecules are therefore removed. To have sufficient unique molecules for good sequencing coverage of your targets, successful captures DEPEND on the input of sufficiently complex libraries. For this reason, for libraries with a significant non-target component (e.g., ancient, forensic, or environmental samples), and especially for WGE captures with a very large full nuclear genome target size, we strongly recommend maximizing the target component in each capture by using as much input library as possible (up to 2 µg+), and consider two rounds of capture for higher percentage of reads on-target.

For best results, it is recommended that only amplified (non-PCR-free) NGS libraries are used for target capture. This provides multiple copies of each starting template molecule, increasing the chance of each individual molecule getting enriched. However if you need more starting material to reach the recommended amount, it is generally preferable to generate more library from fresh genomic DNA or a new batch of indexed library, rather than through extra amplification. This is because while some amplification is good, over-amplification risks reducing the observable complexity of your libraries through the uneven action of PCR bias, as some molecules will become relatively more abundant while others become rare. This is also true for manipulating your libraries after capture: amplify your post-capture libraries the minimum number of cycles necessary to reach the molarity required by your sequencing facility.

We generally do NOT recommend pooling multiple samples per capture reaction for very degraded and/or rare targets (e.g. ancient DNA), or for very large targets (e.g. a WGE baitset targeting a full nuclear genome). In fact, for highly degraded samples, you may achieve better results by performing multiple parallel WGE reactions per sample.

The myBaits WGE product line ONLY offers bait production directly from high quality genomic DNA precursor material. This gDNA must be made physically available to us in order to manufacture a myBaits WGE Custom kit (either extracted in your lab or sourced from a third-party gDNA supplier).

Daicel Arbor Biosciences does have the ability to design and synthesize synthetic customized bait oligo pools via our standard myBaits Custom DNA-Seq, RNA-Seq, and Methyl-Seq products. However the myBaits Custom synthetic oligo approach is generally not an option for targeting entire large nuclear genomes (e.g. from eukaryotic organisms such as plants or animals) due to prohibitively large numbers of baits that would be required. However if you are working with an organism that has a much smaller genome (e.g. one or more bacteria), or are interested in capturing only a portion of a much larger genome, then a myBaits Custom kit is likely the most effective option.

Please contact us with details about your project goals and budget, so we can discuss project options.

We offer complementary myTags Custom (F)ISH probe bioinformatic design service using our proprietary design algorithms for most types of (F)ISH projects. Please contact us with a brief description of your project, including the name of your study species, genomic coordinates, and any additional information.

Our experts will apply our advanced proprietary probe design algorithms to craft a custom probe design for your targeting needs, for any organism or application. Our scientific team has decades of experience designing custom probes for a wide variety of applications, including multi- or single locus/gene localization, chromosome painting, chromosomal indexing/barcoding, haplotyping, and more.

Only the highest specificity regions with minimal background noise and cross-reactivity for premium performance in any downstream experimental application would be selected for the probe design.

And with our transparent and straightforward scientific communication process, you always have full control and ownership over the oligo sequences for each and every myTags Custom probeset.